Copyright 0 1993 by the Genetics Society of America

Unusual Mitochondrial Organization in Cytoplasmic Male Sterile Common Bean and the Nature of Cytoplasmic Reversion to Fertility

H. Janska and S. A. Mackenzie

Department of Agronomy, Purdue University, West Lafayette, Indiana 47907 Manuscript received April 22, 1993 Accepted for publication July 10, 1993

ABSTRACT Spontaneous reversion to pollenfertility and fertility restoration by thenuclear Fr in cytoplasmic male sterile common bean ( L.) are associated with the loss of a large portion of the mitochondrial genome.To understand better themolecular events responsible for this DNA loss, we have constructed a physical mapof the mitochondrial genome of astable fertile revertant line, WPR-3, and thecytoplasmic male sterile line (CMS-Sprite) from which it was derived. This involved a cosmid clone walking strategy with comparative DNA gel blot hybridizations. Mapping data suggested that the simplest model for the structure of theCMS-Sprite genome consists of three autonomous differing only in short, unique regions. The unique region contained on one of these chromosomes is the male sterility-associated 3-kb sequence designated pvs. Based on genomic environments surrounding repeated sequences, we predict that chromosomes can undergo intra- and intermolecular recombination. The mitochondrial genome of the revertant line appeared to contain only two of the three chromosomes; the region containing the pus sequence was absent. Therefore, the process of spontaneouscytoplasmic reversion to fertility likely involves the disappear- ance of an entire mitochondrial . This model is supported by the fact that we detected no evidence of recombination, excision or deletion events within the revertant genome that could account for the loss of a large segment of mitochondrial DNA.

HE mitochondrial genome demonstrates a and WALBOT199 1; NARAYANANet al. 1993) have met T number of features unique to the plant king- with mixed results. Thus, the structure of the mito- dom. The organization of the genome has been the chondrial genome and theexistence of a master mol- subject of investigation for a number of years and still ecule has not been clearly demonstrated. remainssomewhat controversial. It is notclear Recentmapping data in petunia (FOLKERTSand whether the mitochondrial genome exists predomi- HANSON199 l), rice (YAMATOet al. 1992; NARAYANAN nantly as circular or linear DNA molecules (BENDICH et al. 1993) and maize (LEVY,ANDRE and WALBOT and SMITH,1990). The existence of a master chro- 1991) suggest that the structure of the plant mito- mosome, containing the entirety of the genetic in- chondrial genome in some species includes more than formation, has beenproposed butnot yet proven. one autonomousmolecule. The configuration of these Furthermore, little or no information is available re- molecules could not be predicted based on homolo- garding the role of nuclear in the maintenance gous recombinations; consequently,they could not be of the mitochondrial genome organization. referred to as subgenomic molecules. These autono- The most generally accepted model of the mito- mous mitochondrial DNA molecules were designated chondrial genome in higher has consisted of a as chromosomes (LEVY, ANDREand WALBOT1991; multipartite configuration,with a master chromosome ANDREand WALBOT1992; NARAYANANet al. 1993). giving rise to subgenomic circles (LONSDALEet al. Rare recombinationevents across small repeats, or 1988). These subgenomic molecules are presumably site-specific recombination, are considered a mecha- generated by recombinationbetween pairs of re- nism for the generation of these molecules (MANNA peated sequences found on the master chromosome. and BRENNICKE1986; ANDRE,LEVY and WALBOT This model was derived primarily from the mapping 1992). If true, this is of considerable consequence to of overlapping cosmid clones (LONSDALE, HODGEand our understanding of nuclear-mitochondrial interac- FAURON1984; PALMERand SHIELDS1984). Efforts to tions in plants. A mitochondrial genome consisting of test this model by observingintact mitochondrial multiple autonomous chromosomes requires a num- DNA molecules using electron microscopy (KOLOD- ber of essential functions by the nuclear genome to NER and TEWARI1972; SYNENKI,LEVINGS and SHAH maintain a complete mitochondrial genetic comple- 1978; BENDICH1985) or various gel electrophoresis ment for transmission to subsequent generations. procedures (BENDICHand SMITH1990; LEVY, ANDRE The plant mitochondrial genome is generally stable

Genetics 135 869-879 (November, 1993) 870 H. JanskaMackenzie and S. A. as it is inherited generation to generation. One un- Primer sequences were derived from published maize se- usual exception is the process associated with spontan- quences (DAWSON,JONES and LEAVER1984; ISAAC,JONES ousreversion and fertility restoration in the cyto- and LEAVER1985) Other mitochondrial DNA probes used in the study were plasmic male sterility system of common bean (Phas- derived by restriction endonuclease digestion ofcosmid eolus vulgaris L.). Cytoplasmic male sterility (CMS) is DNA, gel electrophoresis, and extraction of the desired a maternally inherited trait resulting in the inability restriction fragment from the agarose. DNA extraction was to produce or shed viable pollen. CMS is caused by a accomplished by emulsification of the agarose band in mitochondrial lesion in nearly allcases examined phenol, freezing at -20", thawing and refreezing, followed by high speed microfuge centrifugation, chloroform extrac- (HANSON 1991). InCMS common bean, recovery of tion, and ethanol precipitation. pollen fertility can occur spontaneously as the result Mitochondrial DNA preparationand cosmid of a low frequency cytoplasmic reversion event or by construction: Mitochondrial DNA was isolated as previously the introduction of the nuclear fertility restorer gene described (MACKENZIEet al. 1988) using differential centrif- ugation followed by DNase I treatment to purify mitochon- Fr (MACKENZIEet al. 1988). By either means, the dria, an SDS buffer lysis of mitochondria, and CTAB pre- recovery of pollen fertility is accompanied by the cipitation of mitochondrial DNA. To construct a mitochon- disappearance of at least 25 kb from the mitochondrial drial of WPR-3, purified mitochondrial genome (MACKENZIEand CHASE1990). This 25-kb DNA was partially digested with Sau3A and size-fraction- regioncontains a 3-kb transcriptionally active se- ated on asucrose gradient (10-4095). Fractions above 30 kb were pooled, ethanol precipitated, and ligated into BamHI- quence unique to the CMS line and designated pvs digested cosmid vector pWE15 (Stratagene). The DNA (JOHNS et al. 1992). ligation mixture was packaged in vitro using Gigapack I1 The process associated with fertility restorationand Gold (Stratagene) according to manufacturer's directions, spontaneous cytoplasmic reversion provides a useful and then transformed to E. coli strain NM 554. A total of genetic system for the study of plant mitochondrial 21 12 colonies were picked and transferred to microtiter plates for storage. The cosmid library of mitochondrial genome structure andits maintenance. We have con- clones from CMS-Sprite was developed previously in vector structed a physical map of the mitochondrial genome pHC79 (MACKENZIEand CHASE1990). of cytoplasmic fertile revertant line WPR-3 and the Cosmid walking strategy:Identification of DNA cosmid predicted corresponding mapof the cytoplasmic male clones encompassing the region associated with sterilitywas sterile line CMS-Sprite to characterize mitochondrial described previously (MACKENZIEand CHASE1990). In CMS-Sprite, a 4.0-kb PstI fragment contains the point of events associated with the reversionphenomenon. divergence between the sterility-associated sequence and the Here we present the results of our mapping analysis sequences repeated elsewhere in the of CMS-Sprite as well as a model forthe mitochondrialgenome and revertant WPR-3. This fragment was usedas a first changes that occur during thefertility reversion proc- probe to screen the revertant WPR-3 cosmid library. All ess. selected clones, after digestion with PstI, showed one config- uration (data not shown). From these initial clones, bidirec- tioncosmid walking was accomplishedusing end-specific MATERIALS AND METHODS RNA probes to select 14 overlapping cosmids at each step. The pWE 15 vector allows the generation of end-specific Plant materials: The cytoplasmic male sterile line of P. RNA probes for each cosmid insert. These RNA probes vulgaris used in the study was derived from the cross of initiate atthe T3 and T7 promotors within the vector GO8063 X Sprite, followed by 16 backcrosses using Sprite (procedure as per manufacturer's instructions). PstI restric- as recurrent pollinator. Fertile accession line GO8063 con- tion maps were constructed for allselected clones. This tains a sterility-inducing cytoplasm (SINGH,WHITE and Gu- allowed the identification of 20 overlapping cosmid clones TIERREZ 1980; MACKENZIE1991). The derived CMS line is that cover the entire WPR-3 mitochondrial genome. To designated CMS-Sprite. The fertile cytoplasmic revertant eliminate the possibility of recombinations during the clon- line WPR-3 was selected from a single seed-bearing pod on ing process, all selected cosmid clones were hybridized to CMS-Sprite. The fully male-fertile line was then selfed over blotted total mitochondrial DNA from WPR-3. five generations to ensure phenotypic stability. Restriction endonuclease analysis, agarose gel electro- Mitochondrial DNA probes: Clones for mitochondrial phoresis and DNA blotting:The cosmid mapping involved ribosomal DNA genes rrn26 from maize (4-kb PvuII; DALE, restriction endonuclease analysis using PstI (New England DUESINGand KEENE 1984) and rrn5/18 from Zea diploper- Biolabs), XhoI (Promega), and Sal1 (New England Biolabs) ennis (1.1-lb HindIII/BglII;GWYNN et al. 1987), ribosomal predominantly, with more refined mapping of some regions subunit rps13, ATPase subunit 9 (atp9) from to- using EcoRI (Promega). All reaction conditions were accord- bacco (0.3-kb EcoRI/BamHI; BLAND,LEVINGS and MATZIN- ing to manufacturer's instructions. GER 1986), maize atpA and NADH-ubiquinone oxidoreduc- Agarose gel electrophoresis in 0.8% agarose, using 1X tase subunit 1 (nadl) from tobacco (1.5-kb BamHIIPstI; TPE buffer, and DNA gel blotting were as described by BLAND,LEVINGS and MATZINGER1986) were supplied by MCNAY,CHOUREY and PRING(1 984) except that Hybond- C. S. LEVINGS;maize cytochrome c oxidase subunit Ill N nylon membrane (0.45 pm, Amersham) was used and (coxZZ1) was supplied by W. HAUSWIRTH,and sorghum ATP- DNA was affixed to the membrane by baking for 2 hr (90") ase subunit 6 (atp6) and cytochrome c oxidase subunit I1 under vacuum. (coxZZ) were supplied by D. R. PRING.Probes for cytochrome DNAprobe preparation and gel blot hybridization c oxidase subunit I (coxZ) and cytochrome b (cob) were analysis: DNA fragments were labeled using the random derived by polymerase chain reaction amplifications using priming method (FEINBERGand VOGELSTEIN1983). Labeled WPR-3 common bean mitochondrial DNAas template. DNA probes were hybridized to DNA gel blotsat 60".Blots Mitochondrial Genome of CMS bean 87 1 A C

probe 1 probe 2 FIGURE1 .-Genomic environ- . kb ' kb 0 e 23.25 (ae. be. ce) ments surrounding the repeat R1. (A) e 8.8 (a-d) Mapping branch points aroundthe * 410.2(C) 1 6 1.2 (b-d. c-d) repeat R 1. Lowercase letters indicate -9.5 (b) different flanking regions, numbered arrows represent hybridization probes. Probe 1: 10.2-kb PstI frag- ment derived from flanking sequence c. Probe 2: atp9 coding region. The approximate locations of atpA and PstI alp9 are shown. (B) Restriction maps -size of products of recombination through 8.8kb the repeat R1. Numbers at the right of the map correspond to the sizes of 24kb PstI fragments containing the repeat. (C) Hybridization experiment using 7.2kb probes 1 and 2 to Pstldigested mito- chondrial DNA from WPR-3 fertile 22kb revertant. The sizes of the hybridizing 1.2kb fragments are indicated at the right. The lowercase letters in parentheses 22kb represent the different combinations of flanking sequences.

were washed twice in 3X SSC, 0.1% SDS at 65" and once with only two to the otherside. As a consequence, the in 0.3X SSC at 65" for 15 min each wash. Autoradiography repeatR1 is present insix genomic environments. was carried outusing X-Omat film(Kodak) with intensifying screens (Picker Intl.). The additional b/c branching point could be a product of a small recombination repeat close to R1, but we RESULTS could not find cosmids containing the four environ- ments predicted around this second small repeat. Fig- Sites of recombination within the mitochondrial ure 1C presents the results of hybridization to a gel genome: Two major families of recombinationally blot of PstIdigested mitochondrial DNA using part active repeats, R1 andR2, were identified in the of the repeat R1 as probe. Restriction enzyme PstI mitochondrial genome of fertile revertant WPR-3. A digests outside of the repeat but within the common repeat was defined as a sequencethat mapped to more b/c flanking region. Therefore, combinations b-e and than one regionof the genome,with recombinational c-e are not distinguished by PstI and both give rise to activity inferred from the presence of multiple com- a 23-kb fragment. Similarly and are not distin- binations of sequences flankingthe repeat.Additional b-d c-d repeated sequences were detected, but with no evi- guished, and give rise to 7.2-kb fragments. Four hy- dence of active recombination. bridization bands are expected, but only three bands Cosmid clones representing six configurations are observed in the figure because of co-migration of around repeat R1 were isolated using a DNA probe 25- and 23-kb fragments under these gel electropho- containing the repeat. These six identified genomic resis conditions. environments are diagrammed in Figure 1. The max- Repeat R2 is also present in six genomic environ- imum fragmentlength shared among all is 5kb, ments and is approximately 4.5 kb in size (Figure 2). suggesting that the R1 repeat is 5 kb or smaller in Two different flanking sequences were identified on size. However, the region of similarity is extended on one endof the repeat. The other end of the repeat is one side of the repeatbetween two flanking sequences flanked by three different configurations,with two of designated b and c in Figure 1. The homology between them sharing an additional 7.5 kb in common. The these sequences is demonstrated by DNA gel blot presence of a second point of divergence (b/c)to one hybridization in Figure 1C. The internal segment of side of R2 was verified by restriction mapping and homology between b and c is less than 1.5 kb, based hybridization experiments similar to those described on restrictionmapping. This additionaldivergence for R1 (data not shown). These hybridization experi- point results in three configurations to one side of R 1 ments also revealed that a part of the segment shared 872 H. Janska and S. A. Mackenzie A C A physical map of the WPR-3 mitochondrial ge- -..- nome: Using overlapping cosmid clone analysis, we kb have constructed a physical map of the mitochondrial genome of revertant line WPR-3 as described in MA- TERIALS AND METHODS. Figure3 presents alinear representation of the physical map with an approxi- - 4-9 (a-d) mate length of 4 17 kb. The repeats R1 and R2 are - 4-7 @-e) each present at three sites. Two copies are in direct orientation and the third in inverted orientation. Six environments surrounding a repeatedsequence requiresthat two repeatconfigurations share one flanking environment. Thisis the case for both repeats R1 and R2. Therefore, to place these repeats onto a single circular map requiresthe introduction of a large duplication. Based on cosmid mapping data presented, B Sal1 the predicted master mitochondrial chromosome in WPR-3 would contain a 246-kbduplication and would a produce a 673-kb molecule. SE~E pips a u :I 11 11 e 7kb An alternate model of the WPR-3 mitochondrial P SEP !E Pi ES genome would predict two autonomous chromosomes b I \ If I I I I d 15kb able to undergo intra- and inter-molecular recombi- P SEP !E PiPS nations (Figure 4A). The region that would be dupli- b I \ If I I! 11 e 13kb P !E S EP Pi ES cated in a master chromosome model is represented EL \ I/ I I! I I d 15kb on both chromosomes as the bold line. Each chromo- P S EP :E PiPS some contains aunique region not present on the c1 I 11 I I! I I e 13kb other(dotted line). Hybridization of WPR-3 total mitochondrial DNA with a radiolabelled clone span- ningunique and duplicated regions of the 257-kb chromosome supports amodel of both duplicated and FIGURE 2.-Genomic environments flanking the repeat R2. (A) unique regions within the genome (Figure 4B). Re- Mapping branch points around the repeat R2. The lowercase letters striction fragments derived fromthe region unique to indicate different flanking regions. The arrow represents the loca- the 257-kb chromosome are present in lower copy tion of probe 3, a l O-kb Psll fragment containing R2.(B) Restriction number than those fragments that are duplicated. maps of the products of recombination through the repeat R2. Both inter-and intramolecular recombination is Numbers at the right indicate the sized ofSalI fragments containing the repeat. (C) Hybridization of probe 3. containing R2, to Sall- predicted between the chromosomes diagrammed in digested mitochondrial DNA from revertant WPR-3. The sizes of Figure 4A. The environments surrounding R1 and the hybridizing restriction fragments are indicated to the right. R2 repeats as presented in the diagram were chosen The lowercase letters in parentheses represent the different com- arbitrarily. All recombinational forms of the repeat binations of flanking sequences. R2 can be explained by intramolecular recombina- between the R2 b and c flanking sequencesis repeated tions between R2 repeats that are in inverted orien- at another site in the WPR-3 genome. This repeated tation. This would produce an inversion of the inter- segment is less than 1 kb in size; however, we have no vening sequence. As a result, different forms of the evidence that this small repeat is recombinationally 394- and 257-kb chromosomesare predicted to occur active. with respect to environments surrounding R2. Intra- We were unable to identify a restriction enzyme molecular recombinations between inverted forms of that would allowus to distinguish the six genomic R1 account for five of the six identified configura- environments around the repeat R2. Digestion with tions. The remainingconfiguration around R1 re- Sal1 produces only four fragments spanning R2 be- quires recombination between the two chromosomes. cause the restriction site is within the b/c divergence In this process, an intermediate of master chromo- point (Figure 2). Combinations b-d and c-d are repre- some size would be formed. Subsequent recombina- sented as 15-kb Sall fragments and b-e and c-e are tion at R2 repeats would then be predicted to produce represented as 13-kb Sal1 fragments. Restriction map- two new circular molecules of 25 1 and 400 kb in size. ping data were verified by hybridization with probes The sequence present between R1 and R2 repeats on encompassing the R2 repeat (Figure 2C) and flanking the 394-kb chromosome in Figure 4A (represented sequences (data not shown) to SalI-digested total mi- by a fine line) can be exchanged between chromo- tochondrial DNA. somes as aresult of this intermolecular recombination. Mitochondrial Genomeof CMS bean 873

0 10 20 30 40 50 kb I I I I I I ,R, I 1 I 1 1 1 PII L

cab ! rrn IWm5

P ~r1 I 1 I II SI II I I I I I I I I

FIGURE3.-A physical map of the mitochondrial genome of fertile revertant WPR-3. Restriction sites are based on digestion with enzymes Psfl (P), Sal1 (S) and XhoI (X). Boxes above the map indicate the locations of recombinationally active repeated sequences. The orientation of the recombination repeats is shown by an arrow. The ends of the map cannot be linked to one another but can be connected to corresponding regions within the map. The left flanking sequence of the repeat R 1, located at the top of the map, can be connected to the left of the R1 repeat adjacent to atpA or to the right of the other R1 repeat. The repeat R2, at the bottom of the map, can be linked to the right flanking sequence of the first R2 repeat or to the left flank of the second R2 repeat. The stippled region contained between vertical dashed lines is present on a 210-kb chromosome in the CMS-Sprite line. The approximate locations of all mitochondrial genes mapped to date are indicated. Placement of mitochondrial genes: The mitochon- of the multiple atp9 environments on geneexpression drialgenes indicated onthe map were placed by is a question for future investigations. hybridization to PstI digested DNA of cosmid clones Comparison of the mitochondrial genome config- spanning theentire mitochondrialgenome. Most urations in CMS-Sprite and WPR-3: A general map genes were present in only one location. However, an of the region encompassing the sterility-associated pvs sequence in CMS-Sprite was presented previously internal region (exon b) of nadl hybridized to two (MACKENZIEand CHASE1990). The pus sequence is PstI fragments separated by approximately 50 kb. We located between sequences thatare repeated else- observed a difference in intensity of these two bands where in the CMS-Sprite mitochondrial genome. when the nadl probe was hybridized to total mito- Theserepeated sequences are also present in the chondrial DNA, suggesting that at least a portion of WPR-3 revertant line. The precise junctions between the nadl gene may bepresent at two sites in the pus unique and repeated sequences were reported by genome. The atp9 gene was located within the R1 CHASEand ORTEGA 992). (1 repeat, resulting in hybridization to three PstI frag- To test for other differences in mitochondrial ge- ments in the genome (Figure1). The influence, if any, nome organization between CMS-Sprite and WPR-3, a74 H. Janska and S. A. Mackenzie

FIGURE4.-(A) Dicircular model A B of the mitochondrial genome in fer- -L kb tile revertant WPR-3.Boxes repre- cox III Sent recombinationally active re- peats, with arrows indicating repeat 4- 8.8 orientation. The region shared by v both chromosomes is indicated by the bold line. The dotted line on each cox II 394 kb m + 5.2 circle designates the regions unique to each chromosome. The sequence represented by a fine line can be ex- changed between chromosomes as a result of intermolecular recombina- tion. Approximate locations of mito- chondrial genes are indicated. (B) PstIdigested mitochondrial DNA from revertant WPR-3 hybridized withcosmid 1H5. This clone over- laps the unique and duplicated re- gions flanking repeat R1 on the 257- kb chromosome. The 5.2-kb band represents a fragment that is re- peated on both chromosomes while the 8.8-kb fragment is unique to the 257-kb chromosome. clones spanning the entire mitochondrial genome of A WPR-3 were hybridized to total mitochondrial DNA preparations from the CMS and revertant lines. No differences were detected between the two lines ex- cept at the region encompassing the pvs sequence. Furthermore, no regions unique to the WPR-3 rev- ertant line were found. These results were obtained using comparative digestions withPstI, SalI, and XhoI. They suggest thatthe mitochondrial genomes of B CMS-Sprite and WPR-3 are colinear, with CMS-Sprite pmbe 4 containing only a single addition of the unique 3-kb pus sequence. The genomic environments flanking the pvs se- quence: The sequences flanking the sterility-associ- ated pus region are present in both CMS-Sprite and WPR-3. Vertical dashed linesin Figure 3 indicate the CMS-Sprile WPR-3 junction points between the pus region and the map of WPR-3.The pus sequence lies immediatelyadjacent FIGURE5.-Extra mapping branch point close to the R1 repeat in the CMS-Sprite genome. (A) Mapping branch points around the to the atpA gene and close to repeat R1. Figure 5A repeat R1 in CMS-Sprite. The lowercase letters indicate different indicates the position ofpus (flanking regionf) relative flanking regions and are the same as Figure 1 with the exception to the otherbranching points surrounding repeat R 1. of the f branch unique to the CMS line. Probe 4 represents a 2.8- The region encompassing pus forms an additional kb EcoRI fragment from CMSSprite. (B) Hybridization of probe 4 to EcoRIdigested mitochondrial DNA from CMS-Sprite and rev- branch point around R1 in CMS-Sprite that is not ertant WPR-3. present in WPR-3.A DNAprobe containing this extra divergence point (probe 4) was hybridized to EcoRI- library but only two from the WPR-3 library. Figure digested mitochondrial DNA from CMS-Sprite and 6A illustrates the relationship between these configu- WPR-3. The resulting hybridization pattern verifies rations. The sequence common to all configurations the presence of this extra configuration in the CMS- is approximately 19 kb and includes R1. The four Sprite genome (Figure 5B). configurations detected usingprobes 4 and 5 are We screened the CMS-Sprite and WPR-3 cosmid presented in Figure 6B. Results of these experiments libraries with the probe 4 (Figure 5) and a fragment indicate that the pus sequence is present in two ge- spanning repeat R1 plus flanking sequence d (probe nomic environments in the sterile line, demonstrated 5, Figure 6). Four classes of clones which hybridized in Figures 6 and 7, with both lost upon spontaneous to both probes were obtained from the CMS-Sprite reversion to fertility. Mitochondrial Genome of CMS bean 875

A R1 apA 7 pvs C afpA \I afD9 /d I I \t yR---,' I ,"-!-; I 10.2 141 6 I 4.5 1 Pal \ I 7 I 6.1 1 1.4 I Sal1 I ;6.3 11.31 I I 5.2 I 6.5 6.6 I Xhol f II FIGURE7.-Regions surrounding the pus sterility-associated se- quence in CMS-Sprite. The pus segment liesbetween sequences C that are repeated elsewhere in the genome of CMS-Sprite. To one side of pus resides a recombinationally active RI repeat. C of the pus sequence(Figure 7); we examinedthe f region flanking the pus sequence to the opposite side f for evidence of a second repeat. If such a repeat were present, we would expect to observe a novel junction fragment in the revertant WPR-3. Similarly, if the pus sequence were lost as the result of a site-specific dele- tion or excision event, we should also observe such a novel fragment unique to theWPR-3 line. CMS-Sprite B cosmid clones spanning over 37 kb onthe side of pus opposite to R 1 were hybridized to total mitochondrial DNA from CMS-Sprite and WPR-3 revertant lines. Individual DNA samples were digested with PstI, SalI, XhoI, EcoRI, DraI, KpnI and SmaI. Comparison of hybridization patterns for the seven different restric- tion patternsrevealed no DNA polymorphism specific to WPR-3 within 37 kb of the pus sequence. Putative structure of the CMS-Sprite mitochon- 1234 1234 I234 drialgenome: DNAgel blot hybridization experi- FIGURE6.-The genomic environments flanking the pus se- ments demonstrated thatall restriction fragments de- quence. (A) Restriction map of four cosmid clones encompassing tected in WPR-3, using PstI, XhoI and Sal1 digestions, the repeat R 1. All of these configurations are present in the CMS- were also present within the CMS-Sprite mitochon- Sprite line, but only two are present in WPR-3 (c-d, c-e). Lowercase letters designate the different flanking sequences. The numbered drial genome. In addition, the CMS-Sprite mitochon- arrows represent probes. Probe 4: 2.8-kb EcoRI fragment from drial genome contained fragments not presentwithin CMS-Sprite; probe 5: 20-kb PstI fragment spanning the repeat R1 the WPR-3 revertant. The simplest model to account and flanking sequence e. (B) Identification of four cosmid configu- forthese data would proposethat the CMS-Sprite rations that hybridized to both probes 4 and 5. Lane 1 represents the c-d configuration, lane 2 (c-e), lane 3 cf-e) and lane 4 cf-d). EcoRI mitochondrialgenome contains the two mitochon- profiles fractionated through 0.7% agarose are shown on the left drialchromosomes present within WPR-3 plus an with corresponding autoradiographs from DNA gel blot hybridi- additional mitochondrial chromosome containing the zation experiments at right. Hybridization with probe 4 produced pvs sequence. This extra chromosome in CMS-Sprite two bands corresponding to flanking sequences c andf. Hybridiza- would be colinear with the region presented between tion with probe 5 produced three bands corresponding to flanking sequences e, with one additional band corresponding to region d. vertical dashed lines in Figure 3. These map break- Differences in hybridization intensities reflect gel loading differ- points are linked by the pus sequence. The size of the ences. predicted chromosome is 2 10 kb (Figure 8). Repeats R1 and R2 contained on the pvs chromosome can The proximity of a recombinationally active repeat undergointermolecular recombinations with corre- to a unique sterility-associated sequence has been ob- sponding repeats on the 394- and 257-kb chromo- served in other CMS systems (FAURON, HAVLIK and somes. As a consequence of intermolecular recombi- BRETTELL1990; FOLKERTSand HANSON 1991;FAU- nation between the 394- and 2 10-kb chromosomes, RON et al. 1992). In CMS-T maize, it has been sug- the pus sequence could be present on an alternative gested that recombination by two families of repeats, 204-kb molecule (Figure 9). These 204- and 2 10-kb followed by the selective elimination of recombination pus-containing molecules account for thetwo genomic products containing the T-urfl3 sequence, would ac- environments of pus found in the CMS line. count for theobserved loss of the sterility sequence in To evaluate the relative copy number of molecules tissue culture-induced revertants to fertility (FAURON, containing the pus sequence, we compared copy num- HAVLIKand BRETTELL1990). In CMS-Sprite, we ber of a fragment that encompasses the breakpoint found that a copy of the R1 repeat resides to one side between common (atpA region) and unique sequences 876 H. Janska and S. A. Mackenzie

FIGURE 9.-Two different pus-containing circles are predicted within the CMS-Sprite genome based on restriction mapping data. The 204-kb moleculeis predicted to arise by intermolecular recom- I bination between the 2 10- and 394-kb chromosomes diagrammed in Figure 8. As a consequence, the sequence between the R1 and R2 repeats, represented by a tine line, is exchanged between chro- - mosomes. FIGURE8.-Proposed model of the CMS-Sprite mitochondrial ated by recombination. Analysis of the mitochondrial genome. The CMS-Sprite genome contains two chromosomes iden- genome configuration of revertant WPR-3 indicates tical to those mapped within revertant WPR-3 plus one additional that the process of reversion likely involves the loss of chromosome containing the sterility-associated pus sequence. This extra chromosome is colinear with a part of the 394-kb molecule the pus-containing chromosomes from the CMS ge- (stippled region), with breakpoints linked together by the pus seg- nome (Figure 10). This model is supported by the fact ment. These breakpoints are indicated in Figure 3 by vertical lines. that we detected no evidence near the pus sequence Boxes represent recombination repeats with arrows indicating ori- of recombination, excision or deletion events that entation. Approximate locations of mitochondrial genes are indi- cated. would be necessary if less than an entire chromosome were deleted from the genome. on the 210- and 394-kb chromosomes (see Figure 8). In predicted mitochondrial genome configurations The probe used was internal to both segments and of CMS and revertant lines, a large portion of each produced a 2.8-kb EcoRI fragment unique to the pus autonomous molecule is shared. Therefore, the mol- region and a 3.6-kb EcoRI fragment unique to the ecules are distinguished by only short regions unique 394-kb molecule. Densitometry scans of multiple au- to each. Homologous recombination events are as- toradiographs produced by hybridization indicated a sumed to account for the various arrangements sur- ratio of 1:1.2 (2.8:3.6 kb) (data not shown). Because rounding repeats R1 and R2. It is also possible that of the predicted complexity of the genome due to homologous recombination occurs across the long recombination, hybridization analysis does not indi- stretches of sequence shared by each chromosome, cate the ratio of one predicted molecule relative to although our study could not test for this. Conse- another but rather,indicates the relative copy number quently, the data presented here cannot exclude the of the two sequences monitored. These results show possibility that the predicted mitochondrial molecules that the sterility-associated pus sequence is not con- interconvert to form a master circle. However, pro- tained on a substoichiometric molecule. posal of a master chromosome would necessitate the triplication of a segment over 200 kb in size onto a DISCUSSION single molecule in the CMS genome. Spontaneous cytoplasmic reversion to fertility in The possibility of morethan one autonomously CMS common bean is associated with the loss of at replicating form within the plant mitochondrial ge- least 25 kb encompassing the sterility-associated pus nome has been raised previously. Coexistence of two sequence (MACKENZIEand CHASE1990). To charac- distinct mitochondrial chromosomes based on cosmid terize better the molecular events associated with re- mappingdata was first proposed inCMS petunia version, we have constructed physical maps of the (FOLKERTSand HANSON1991). In the maize BMS mitochondrialgenomes of afertile revertant line, cultivar, use ofpulsed field gel electrophoresis allowed WPR-3, and the CMS line from which it was derived. the mapping of a 120-kb chromosome that does not Based on cosmid mapping and mitochondrial DNA contain any large repeats, suggesting that it cannot be gel blot hybridization data, thesimplest model for the generated by recombination from a master circle and structure of the CMS-Sprite mitochondrial genome must be maintained autonomously (LEVY,ANDRE and consists of three chromosomes able to undergo intra- WALBOT199 1). The genome organizationof multiple and intermolecular recombination. The pus sequence autonomous chromosomes has also been suggested in is present in two differentgenomic environments, the development of a physical map of the rice mito- suggesting that the pus sequence is contained on at chondrial genome (YAMATOet al. 1992; NARAYANAN least two mitochondrial DNA molecules, one gener- et al. 1993). Therefore, the predicted configuration Mitochondrial Genome of CMS bean 877 CMS-Sprite- REVERTANT WPR-3

REVERSION

n

257 kb

UD6

cox I1

6

UP 9 k FIGURE 10.-Proposed model of reversion to fertility in CMS P. vulgczris. Based on mapping data, we propose that spontaneous reversion to fertility involves the loss of the ps-containing chromosomesfrom the CMS-Sprite mitochondrial genome. of the bean mitochondrial genome was not altogether of common bean have been reported previously and surprising. were taken into account during the interpretation of The proposed model of the genome in common our mapping data. Although small supercoiled DNA bean is consistent with six genomic environments sur- molecules have been observed in mitochondrial prep- roundingrepeats R1 and R2. Analysis of cosmid arationsfrom common bean (DALE,DUESING and clones containing repeat sequences and hybridization KEENE 1981), we did not observe these molecules in of these cosmids to gel blots of total mitochondrial mitochondrial DNA preparations fromCMS-Sprite or DNA preparations showed that there is an unequal WPR-3. This could reflect differences in mitochon- number of flankingsequences to each side of the drial DNA isolation procedures or differences in plant repeats. Similar observations were made in petunia genotypes. Consequently we were unable to account (FOLKERTSand HANSON 1991)and rice (YAMATO et for the genetic organizationof these supercoiled mol- al. 1992). In both cases, it was suggested thatthe ecules. missing environments arounda repeat were lost from Using three restriction enzyme digestion profiles, the genome. In rice,one sequence flanking repeat R1 Khairallah, Adams and Sears (1991) estimated the was missing in the tissue cultured A-58CMS line while mitochondrial genome size of P. vulgaris to be close it was present in the cultured Chinsurah BoroI1 line. to 450 kb. Based on the linearrepresentations of Similarly, nine genomic environments surrounding a physical mapping data, the mitochondrial genomes of repeat were found in the petunia fertile line 3704, WPR-3 and CMS-Sprite are 417 and 420 kb in size, but only six flanking environments were identified in respectively. These estimates agree with the earlier the sterile line 3688. Investigation of the progenitor study. However, if we sum fragments from both chro- lines to CMS-Sprite may allow us to characterize the mosomes in WPR-3 or thethree chromosomes in events leading to the unusual genome organization CMS-Sprite in our calculations, the sizeof the ge- observed in bean. nomes would increase substantially. This discrepancy Analysis of the progenitors to CMS-Sprite might between earlier estimates and the size of the genomes also answer questions about the nature of additional based on physical mapping data can be explained by branching points identified to one side of both R1 the fact that determination of genome size using re- and R2 repeats. At least two interpretations of these striction patterns prevents accurate determination of branch points are possible. These divergence points fragment copy number. According to our multiple could be the products of recombination at small re- chromosome model, regions presentat onecopy num- peats nearby to the larger, more active repeats R1 ber per genome would appear in digestion profiles as and R2. Alternatively, R1 and R2 may exist in both submolar bands. full length andtruncated forms. We are currently What is most unusual about our observations in the investigating the structure andlocations of repeats R1 CMS bean system is suggestion of the loss of an entire and R2 in the reported progenitor lines NEP-2 and mitochondrial chromosome upon reversion to fertil- POP to better understand theorigins of this unusual ity. We have detectedno differences between the genome configuration. structure of the mitochondrialgenome of sponta- Studies of the mitochondrial genome configuration neous revertant lines and fertile lines that have been 878 H. Janska and S. A. Mackenzie restored by the nuclear gene Fr (MACKENZIEet al. Fr as afactor in mitochondrialchromosome copy 1988; MACKENZIEand CHASE1990). This result sug- number control. Introduction of Fr would then lead gests thatthe mitochondrialalterations associated to areduction in replication of the pus-containing with the process of reversion are identical to those chromosome relative to the remainderof the genome. effected by the nuclear restorer gene. We selected However, it is more difficult to visualize the role of fertile revertant WPR-3 for this study, rather than a replication rate in effecting a spontaneous eventsuch fertile restored line, because WPR-3 and CMS-Sprite as cytoplasmic reversion, particularly when the chro- are genetically isonuclear, eliminating the possibility mosome deleted is not substoichiometric. of unrelatedmitochondrial changes resulting from An alternative model for the reversion and fertility variation in nuclear genotype that may accompany the restoration process would involve the unequal sorting introduction of Fr. of mitochondria leading to the random loss or selec- In maize CMS-T, tissue culture-induced cytoplasmic tive elimination of those mitochondria containing the reversion to fertility is correlated with there- pus sequence. This model would presuppose the exist- arrangement of the mitochondrial genome by recom- ence of a heterogeneous population of mitochondria bination (FAURON, HAVLIKand BRETTELL1990; FAU- in the CMS line, those containingthe pus chromosome RON et al. 1992). We have no evidence of recombina- and those withpus absent. Aberrant mitochondrial tion in the mitochondrial alteration associated with transmission during mitosis and zygote formation has reversion inCMS bean, based on the fact that we been observed in a number of mutants of Saccharo- detected no products of recombination in the rever- myces cereuisiae (DUCHER1982; MCCONNELLet al. tant line. Comparison of DNA hybridization patterns 1990; DIFFLEYand STILLMAN1991; ZWEIFEL and between the WPR-3 and CMS-Sprite genomes, using FANCMAN 1991;JONES and FANCMAN1992; AZPIROZ three restriction enzymes for the entire genome plus and BUTOW1993; CHENand CLARK-WALKER 1993). four additional enzymes in the region encompassing In nearly all cases, mitochondrial sortingand selection pus, revealed no fragments unique to the revertant. are under nuclear gene control. In , heter- We have not eliminated the possibility that both prod- oplasmy is also associated with changes in frequency ucts of recombination are undetectable using this of mitochondrial fusion. The phenomenon of mito- approach. Interestingly, the model accounting for mi- chondrial fusion in the Physarum system is regulated tochondrial rearrangementsin revertants of themaize by nuclear genotype (KAWANOet al. 1993). CMS-T system assumes that the intramolecular re- A mechanism of unequal mitochondrial sorting or combination events are followed by the spontaneous selection would account for the spontaneous nature loss or elimination of two of the four recombination of cytoplasmic reversion events;one could envision an products. The process by which this DNA loss occurs unequal partitioning of mitochondria during gamete may be similar tothat involved in the CMS bean formation leading topollen and/or egg cells with few system. or no pus-containing mitochondria. A similar result The mitochondrial genome of CMS petunia bears by the introduction of nuclear fertility restorer gene Fr would suggest that Fr participates as anuclear important similarities to that of CMS bean. The CMS factor in the direction of the cytoplasmic sorting proc- petunia line 3688 consists of two circular DNA mol- ess. We are currently using both genetic and molecu- ecules differing at only one site. One of the chromo- lar approaches totest these alternative models. somes contains the CMS-associated pcf sequence. The elimination of this molecule would presumably result The authors wish to express thanks to STANGELVIN, SHICHUAN in recovery of fertility. However, spontaneous rever- HE and ANNALYZNIK for their critical reviews of the manuscript. sion to fertility is not observed. This is likely due to This work was supported in part byU.S. Department of Agriculture genetic linkage between the pcfsequence and theonly grant 90-37262-5652 and National Science Foundation grant 9118937-MCB to S.M. This is journal series no. 13767 from the copy of nad3 and rps 12 genes ( FOLKERTSand HANSON Indiana Agricultural Experiment Station. 1991). The elimination of the pcf-containing chro- mosome would result in loss of not only the sterility- LITERATURECITED associated sequence but two genes essential for mito- chondrial function.In thecase of CMS bean, however, ANDREC., A. LEVYand V. WALBOT,1992 Small repeated se- quences and the structure of plant mitochondrial genomes. the pus-containing chromosome appears to be dispen- Trends Genet. 8: 128-132. sible, with all sequences linked to pus repeated else- AZPIROZ,R., and R. Bmow, 1993 Patterns of mitochondrial where in the genome. sorting in yeast zygotes. Mol. Bioi. 4 21-36. The loss of amitochondrial chromosome might BENDICH,A. J., 1985 Plant mitochondrial DNA: unusual variation occur by at least two mechanisms. One possibility is on a common theme, pp. 111-138 in Genetic Flux in Plants, edited by B. HOHNand E. S. DENNIS.Springer-Verlag, Wien. by differential rates of replication of individual mito- BENDICH,A., and S. SMITH, 1990 Moving pictures and pulsed- chondrial chromosomes. This model is feasible when field gel electrophoresis show linear DNA molecules from speculating on a role for nuclear fertility restorer gene and mitochondria. Curr. Genet. 17: 421-425. Mitochondrial Genome of CMS bean 879

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